Abstract The meniscus plays a vital role in healthy knee function. However, given the centrality of this tissue in load transfer and the demanding physical environment, injury is common and healing in adults is limited. The current lack of regenerative solutions for knee meniscus injury arises, in part, from the significant gap in technology that can be used to repair the dense and complex tissue structure of the meniscus. Particularly challenging is regenerating the distinct inner and outer zones of tissue, which have distinct composition and mechanical properties to enable effective load bearing. While scaffolds that mimic the bulk geometry of meniscus are being introduced clinically to promote the repair/regeneration of meniscus, these scaffolds do not provide the microscopic, cell-scale cues that are needed to simulate cells to form tissue matching these region- specific attributes. This limitation leads to immature meniscus-like tissue, lacking in load-bearing capacity. It is increasingly well understood that structural properties (e.g., shape and stiffness) of cell-scale structures can influence the cell activity and direct matrix formation. To harness these insights, our team developed the FiberGel system to generate biopolymer-based, cell-scale microfibers, in which individual fibers have a tunable diameter and stiffness. These microfibers can be molded and crosslinked into various shapes to fill meniscus defects, and the internal microfibers can be formed into a random or aligned configuration to mimic the different regions of the native meniscus. Meniscus cells can be directly mixed into the FiberGel paste before crosslinking to produce uniformly cellularized constructs. In this proposal, we optimize this promising material to define conditions that promote formation of inner and outer zone phenotypes and structures. We will determine how the tunable parameters of FiberGel system ? diameter, stiffness and alignment ? impact the biosynthetic activities of meniscus cells through a series of studies designed to refine and optimize matrix formation. To determine how FiberGel-based implants respond to mechanical forces that arise with joint motion, we will also use a custom mechanical bioreactor to apply physiologic load to constructs during their maturation. Our central hypothesis is that there exists an optimal set of microfibers parameters (diameter, stiffness and alignment) for regenerating the inner and outer regions of our meniscus. Successful completion of this work will generate FiberGel formulations that may be used for the clinical repair of the knee meniscus.